How To Get a Perfect Pellet After DNA/RNA Precipitation
If you’re performing DNA/RNA precipitations, you will have read Suzanne’s excellent article on which alcohol to use for precipitating your precious samples (check out some useful info in the comments for that article as well).
Its publication prompted the recall of a useful tip I learned from a post-doc many years ago, one of those ‘magic-hands’ scientists where everything seemed to work first time. This tip of his is on the removal of the 70% ethanol in the final step of the precipitation process.
The usual wisdom is to carefully drain off the ethanol and leave it to either air-dry or place in a speed-vac. The problem is that air-drying can be too slow, while the speed-vac can be too fast meaning that you risk over-drying the pellet making solubilisation more difficult.
So here’s the tip: After draining or pipetting off the 70% ethanol, simply:
- recap the tube
- pulse the centrifuge to bring down the remaining ethanol
- remove this liquid with a pipette and 200ul tip – you can get right alongside the pellet if visible. Leave the tube open as you move to the next sample.
By the time you’ve finished your series of tubes (even if n= 3), the pellets are dry always enough to add diluent immediately. Job done. This saves time and give you samples are just the right degree of “dry”, every time.
Any other tips for ethanol (or even isopropanol!) precipitation would be great to hear. Meanwhile – thanks Michael.
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John Mackay
John is interested in using molecular diagnostics to solve industry issues - working on samples as diverse as wine, truffles, grapevine and human. He also consults on PCR and real-time PCR assay development as technical director for the company dnature.
What I usually do is aspirating the alcohol with a pasteur pipette connected to a water-vacuum-thing. Of course I proceed fairly cautiously so I never lost a pellet. This technique gets you pellets that are almost instantly “dry” without centrifuging again. You also dont have to be permanently “in” the liquid, in my case the negative pressure is enough to actually get rid of the ethanol or isopropanol without touching the bottom of the tube. Should the pellet detach from the tube and swim in, say 50µl, simply flick until either the pellet or the ethanol is separated from the rest on the side of the tube. You can then aspirate the rest easily.
A very similar way there – standard Maniatis I think? But largely replaced with the ’steady pour and flick’ I think
Either method, if you have the pellet-side uppermost in the tube, you can drain the tube with the pipette tip quite safely.
One other suggestion (esp if dissolving pellet in small volume), add the volume of diluent to halfway down the tube above the pellet and let it slip down the side. Nucleic acid will be in a relatively large area rather than just in the small pellet (again, if visible)
These days I only do alcohol precipitation to sterilize DNA before transfections. With silica-based methods ubiquitous, can’t think of very good reasons to do alcohol precipitations anymore. I have always done what’s described above. Don’t have a patience to wait and yes, it works great. Few more tiniest hints:
1. Whenever possible/convenient/purity-matters-more-than-usual, let DNA sit in 70% EtOH for anywhere from 1 hour to o/n. This results in cleaner, faster drying pellet that gives less denatured DNA upon resuspension (the small amount of stuff that hardly even enters gel).
2. When working with large DNAs (say, 50-200 kbp), take care not to over-dry. If anything, it is better to have some solution left in the pellet than dry it to death. Large dry DNA takes forever to hydrate and go onto solution – and pipetting to help is a no-no because it is prone to shearing.
3. After precipitating dirty/semi-dirty DNA material that contains fair amount of protein, spin final resuspended DNA hard and very carefully take sup. Most proteins are denatured irreversibly in 40-50% isopropanol and will pellet again, leaving cleaner DNA in the sup.
So – to let the DNA sit in the 70% ethanol (1 hour or add and spin as often done) or vortex the pellet to more thoroughly wash and de-salt (apparently)?
Def good idea to re-pellet the crud if present – often bits of crud will inhibit a PCR (as I refined during a very rapid method developed to extract plant viroid RNA)
Vortexing should greatly reduce the amount of DNA as a large portion of salt has already been washed away. So you won’t be able to pellet as much DNA as you had before vortexing, it will stay in solution.
This final spin also helps bring DNA that was spread across the back of the tube down into the pellet. I’ve had pellets that appeared after this extra spin that weren’t there just after the 70% Ethanol wash step. In order to get this to work, though, it seems that you have to work quickly – presumably before the DNA dries onto the tube. I spin for 1 minute, but shorter spins may be sufficient for bringing down dispersed samples.
While I also aspirate off the liquids for the earlier steps, after this final spin I draw off any remaining liquid with a gel-loading tip on a P20 or P200, if necessary.